Until now, it was believed that only one form of arginine vasotocin (AVT)-regulated aquaporin (AQP) existed to control water absorption from the ventral skin of semiaquatic anuran amphibians, eg, AQP-rj3(a) in Rana japonica. In the present study, we have identified a novel form of ventral skin-type AQP, AQP-rj3b, in R. japonica by cDNA cloning. The oocyte swelling assay confirmed that AQP-rj3b can facilitate water permeability. Both AQP-rj3a and AQP-rj3b were expressed abundantly in the ventral hindlimb skin and weakly in the ventral pelvic skin. For the hindlimb skin, water permeability was increased in response to AVT, although the hydroosmotic response was not statistically significant in the pelvic skin. Isoproterenol augmented water permeability of the hindlimb skin, and the response was inhibited by propranolol. These events were well correlated with the intracellular trafficking of the AQPs. Immunohistochemistry showed that both AQP-rj3 proteins were translocated from the cytoplasmic pool to the apical membrane of principal cells in the first-reacting cell layer of the hindlimb skin after stimulation with AVT and/or isoproterenol. The type-b AQP was also found in R. (Lithobates) catesbeiana and R. (Pelophylax) nigromaculata. Molecular phylogenetic analysis indicated that the type-a is closely related to ventral skin-type AQPs from aquatic Xenopus, whereas the type-b is closer to the AQPs from terrestrial Bufo and Hyla, suggesting that the AQPs from terrestrial species are not the orthologue of the AQPs from aquatic species. Based on these results, we propose a model for the evolution of cutaneous water-absorbing mechanisms in association with AQPs.